Enzymes may be immobilized by a variety of chemical or physical techniques. Until now, all chemical techniques have involved the modification of the amino acid residues of an enzyme, even though the enzyme may have contained other functional groups which could be employed, e.g., the carbohydrate residues of glycoenzymes. Thus, glycoenzymes such as glucose oxidase or glucoamylase have been immobilized by chemical modification of only their amino acid residues. Recent work on the function of the carbohydrate residues of glucose oxidase has suggested that these sugar moieties are not involved in catalysis, thus it appears that it would be more desirable to covalently bond glycoenzymes to water-insoluble polymers by these catalytically nonessential carbohydrate groups than by amino acid groups, some of which are responsible for substrate binding and catalysis. This invention thus relates broadly to "non-amino acid immobilization" of enzymes.
The modification of glycoproteins with periodates is known in the art. For example, see Biochemical and Biophysical Research Communications, Vol. 31, No. 1, 1968 which teaches the modification of horseradish peroxidase by sodium metaperiodate to form an oxidized product. The author does not discuss the further reaction of this oxidized product to prepare, for example, an immobilized enzyme. Bossard, Annee Biol. 52, 202 (1948) teaches that periodic acid destroys the glucosidase of almonds. Similarly, Maekawa et al., Vol. 29, No. 7, at pages 353-356, Proc. Japan Acad. (1953) show the oxidation of alpha amylase with sodium metaperiodate to form an oxidized product which possesses only a small amount of its original activity. Again, oxidized alpha amylase was not suggested for further reaction nor as a precursor for an immobilized amylase composite.
In Archives of Biochemistry and Biophysics, 103, 515-518, 1963, Pazur et al. disclose periodate oxidation of alpha amylase and glucoamylase to obtain a product which has no loss in enzymatic activity. However, the authors did not suggest that this product could be further reacted to prepare an immobilized enzyme. Pazur et al. (Arch. Biochemistry and Biophysics, 111, 351-357 (1956)) teach the oxidation of the carbohydrate residue of glucose oxidase with sodium metaperiodate. The authors report that the enzyme loses activity after oxidation and do not suggest the further reaction of the oxidized product to prepare, for example, an immobilized glucose oxidase.